The polycation DEAE-dextran treatment of HeLa cells was found to interfere with the production of reovirus, appearance of viral cytopathology, and the induction of cytotoxicity by UV-irradiated reovirus. The data obtained showed that while the polycation pre-treatment of cells enhanced virus adsorption to cells, its addition early during virus adsorption or 2 h after infection markedly interfered with virus production. The interference was decreasingly effective when the polycation was added during the later stages of the infectious cycle. Isopycnic CsCl buoyant density ultracentrifugation analyses of infected cytoplasmic extracts revealed that there were more subviral particles formed in untreated infected cells than there were in polycation-treated cells. In vitro uncoating studies with infected cytoplasmic extracts indicated that the polycation did not interfere with the removal of the outer capsid structure of the complete virus, and uncoating occurred only in the presence of the polycation. Electron microscopical examination revealed significantly fewer virus particles present in polycation-treated infected cells. The accumulated data strongly indicate that interference by DEAE-dextran is an early event involving viral penetration. Corollary studies using other polycations, such as polybrene (hexadimethrine bromide and poly-l-lysine, revealed that while they also enhanced virus adsorption, they did not interfere with reovirus production, appearance of viral cytopathology, and the induction of cytotoxicity by UV-irradiated reovirus.